HIV-1 anti-core neutralizing antibodies that target a conformational epitope within the ALPHA5-helix of GP120

Abstract

Broad neutralizing antibodies directed to epitopes of Human Immunodeficiency Virus, or hiv, especially the preparation and use of highly neutralizing antibodies directed to hiv gp120 envelope protein, in the vaccination and treatment of hiv-infected patients.

Description

FIELD

This invention relates to antibodies directed to epitopes of Human Immunodeficiency Virus, or HIV, especially the preparation and use of highly neutralizing antibodies directed to HIV gp120 envelope protein, in the vaccination and treatment of HIV-infected patients.

BACKGROUND

Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus identified as the human immunodeficiency virus (HIV). The immune response to HIV infection in long-term non-progressors suggests that specific viral immunity may limit infection and the symptoms of disease. Rare HIV infected individuals show broadly neutralizing IgG antibodies in their serum but little is known regarding the specificity and activity of these antibodies despite their potential importance in designing effective vaccines. No single characteristic yet correlates with protective immunity. In animal models, passive transfer of neutralizing antibodies can also contribute to protection against virus challenge. Neutralizing antibody responses can also be developed in HIV-infected individuals and are associated with lower viral loads in long-term non-progressors. Though this neutralizing antibody response is uncommon, it is directed largely against the Env protein of the virus.

A number of immunologic abnormalities have been described in AIDS including abnormalities in B-cell function, abnormal antibody response, defective monocyte cell function, impaired cytokine production, depressed natural killer and cytotoxic cell function, and defective ability of lymphocytes to recognize and respond to soluble antigens. Other immunologic abnormalities associated with AIDS have been reported. Among the more important immunologic defects in patients with AIDS is the depletion of the T4 helper/inducer lymphocyte population.

The HIV env protein has been extensively described, and the amino acid and RNA sequences encoding HIV env from a number of HIV strains are known (Modrow, S. et al., J. Virology 61(2): 570 (1987). The HIV virion is covered by a membrane or envelope derived from the outer membrane of host cells. The membrane contains a population of envelope glycoproteins (gp 160) anchored in the membrane bilayer at their carboxyl terminal region. Each glycoprotein contains two segments. The N-terminal segment, called gp120 by virtue of its relative molecular weight of about 120 kD, protrudes into the aqueous environment surrounding the virion. The C-terminal segment, called gp41, spans the membrane. gp120 and gp 41 are covalently linked by a peptide bond that is particularly susceptible to proteolytic cleavage. McCune et al., EPO Application No. 0 335 635, priority 28 Mar. 88 and references cited therein, herein incorporated by reference.

Several approaches to an AIDS vaccine have been proposed, including inactivated and attenuated virus vaccines, subunit vaccines from virus-infected cells, recombinantly produced viral antigens, vaccines based on synthetic peptides, anti-idiotypic vaccines, and viral carrier-based vaccines, however no vaccination study published to date has provided protection against challenge with virus. Several reviews of HIV vaccine development have been published, e.g. Lasky, (1989), Newmark, (Jun. 23, 1988), and Fauci et al., (1 Mar. 1989).

However, there have been problems including reversion of attenuated virus vaccines; soliciting partial immune response; not eliciting cellular immunity; and the variability and mutability of HIV itself. These problems have frustrated the development of HIV therapies.

Other approaches to HIV therapeutic and prophylactic treatments have included making highly neutralized antibodies for HIV treatment. There have been many years of extensive HIV research in cloning and making monoclonal antibodies by various techniques for targeting CD4 and for neutralizing HIV. These techniques usually involve self-fusion or phage display techniques. A limited number of monoclonal antibodies produced so far are broadly neutralizing to HIV. Among those broadly neutralizing monoclonal antibodies is the phage display antibody, B12. Typically, in making HIV neutralizing antibodies using phage display techniques, random combinations of heavy and light chains are combined and a random pair is selected; in this instance, B12 was shown effective. Monoclonal antibody B12 is a broadly neutralizing antibody which prevents HIV infection in Macaques. Another broadly neutralizing antibody includes 2G12, which, atypically, has a structure which has yet to be seen in any other antibody with three combining sites. The structure of 2G12 has yet to be reproduced.

It has been found that certain people develop antibodies to HIV, which are broadly neutralizing antibodies, which means that their antibodies can neutralize many strains of HIV in their sera. While such patients do not cure their own infection, they are able to inhibit it. When the antibody sees the virus, the virus mutates away from the antibody. These people continue to make antibodies even as the virus keeps mutating. Antibodies can be protective against initial HIV infection in passive transfer experiments in non-human primates and can modulate viral load during infection. Mascola, 2000; Shibata, 1999; Veazey, 2003; Parren, 2001; Mascola, 1999; Trkola, 2005; Wei, 2003; Frost, 2005. Based on these observations, it has been proposed that such antibodies may be important components of a preventative vaccine. Burton, 2004; Mascola, 2007; Karlsson Hedestam, 2008; McMichael, 2006; Zolla-Pazner, 2004.

These Rare HIV infected patients develop high titers of broadly neutralizing antibodies. But, despite intensive study over two decades, only a small number of well-characterized monoclonal antibodies broadly neutralize HIV efficiently in vitro and only a fraction of these prevent infection of non-human primates, as discussed herein, Mascola, 2000; Shibata, 1999, Veazey, 2003; Parren, 2001; Mascola, 1999. At present, broadly neutralizing antibodies from non-progressor patients and/or slow progressor patients have not been made or isolated. It is therefore a continuing need to identify and produce neutralizing antibodies for the development of HIV therapeutics and vaccines.

Citation of the above documents or any references cited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

SUMMARY

The invention described herein provides for neutralizing antibodies to HIV and method of using and making such HIV neutralizing antibodies (“HIV neutralizing antibodies”). In certain embodiments, these HIV neutralizing antibodies can be derived from non-progressor and slow-progressor HIV patients.

This invention is particularly directed to HIV neutralizing antibodies comprising at least one of an antibody, or antigen binding portion thereof, which comprises a binding region binds to an antigenic epitope on gp120, or a portion of the antigenic epitope, wherein the antigenic epitope is on the same face of gp120 as a CD4 binding site. The antigenic epitope may also be on the same face as the binding site for a b12 antibody. An aspect of the invention provides for antibodies, or an antigen binding portion thereof, which binds to a new the antigenic epitope comprises gp120^core, which binds to the same face of gp120 as b12 and CD4.

It is an aspect of the invention to provide for an antibody, or antigen binding portion thereof, comprising a binding region which comprises a CDR3 region comprising at least one of SEQ ID NOs: 1-630, or fragments or derivatives thereof. The antibodies which bind to a CDR3 region further may bind to epitopes, or portions of epitopes, comprising V3, gp41, VL, CD4i or CD4bs. In yet another aspect, the invention provides for a vaccine comprising at least one antibody of the invention and a pharmaceutically acceptable carrier.

Another aspect of the invention provides for a method of inhibiting virus replication or spread to additional host cells or tissues comprising contacting a mammalian cell with at least one antibody of the invention. An aspect of the invention further provides for a method for treating a mammal with infected with HIV administering to said mammal a pharmaceutical composition comprising at least one antibody according to the invention.

It is a further aspect of the invention to provide for a method for vaccinating a mammal from HIV infection. The vaccines or antibody pharmaceutical compositions of this invention may be administered alone or in combination with other HIV antigens, and in one or several immunization doses. It is further an aspect of the invention to vaccinate a mammal by administering epitopes that bind the antibodies, comprising any one of SEQ. ID No. 1-630, or any combination thereof.

The objects of this invention are accomplished by the preparation and administration an HIV antibody preparation which is suitable for administration to a human or non-human primate patient having or at risk of having HIV infection, in an amount and according to an immunization schedule sufficient to induce a protective immune response against HIV.

It is therefore an object of this invention to provide for compositions of broadly neutralizing antibodies that can elicit a protective immune response against HIV infection. It is a further object of this invention to provide methods for preparing and administering such compositions.

These and other objects of some exemplary embodiments will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments without departing from the spirit thereof. Additional features may be understood by referring to the accompanying drawings, which should be read in conjunction with the following detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Anti-gp140 Antibody Cloning.

A) Flow cytometry plots of peripheral blood monocular cells from four HIV patients stained with anti-CD 19 and biotin-gp140. B) Distribution of gp140 binders and non-binders among all antibodies cloned. C) Igκ and Igλ expression among all gp140 binding antibodies. The number in the center of the pie charts indicates total number of antibodies analyzed, each pie slice represents a unique Ig heavy and light chain pair and the size of the slice is proportional to the number of clonal members. Each clonal family shaded throughout and unique antibodies that are not members of clones are not shaded. D) gp140 binding ELISA results for a set of representative antibodies cloned from gp140 binding memory B cells from patient 1 (left), patient 2 (middle), and from IgG B cells that did not bind to gp140 from patient 2 as control (right). Each line represents an individual antibody. One line shows the binding characteristics of the anti-gp140 antibody b12, (Burton, 1991), another line is a negative control antibody mGO53 (Wardemann, 2003). Antibody concentration in μg/ml is on the X-axis and optical density values on the Y-axis.

FIG. 2. Anti-gp140 Antibody Repertoire.

Top line indicates patient number and whether the antibodies bind to gp140. IgGm are previously published IgG memory antibody controls {Tiller, 2007}. Each clone is represented once irrespective of clone size, and somatic variants are not considered. A) IgH repertoire analysis comparing V_H (top) and J_H (bottom). Each slice represents a V_H family or J_H as indicated at right. B) IgH CDR3 positive charges (top, pies) and length (bottom, histograms). Each pie slice or histogram bar is shaded to indicate number of positive charges or amino acids as indicated. C) Igκ repertoire comparing Vκ (top) and Jκ (bottom). Each slice represents a Vκ family or Jκ as indicated. D) Graphs show numbers of mutations per antibody for V_H (left) grouped by patient, Vκ (middle) grouped by patient, and V_H (right) for all antibodies to a specific epitope as indicated. Stars indicate p values ≦0.001.P values were calculated by comparison to the pool of gp140 non-reactive antibodies except those below the lines, which refer to the paired samples. P values for Ig gene repertoire analyses were calculated by 2×5 Fisher's Exact Test and Chi-Square test. Statistical analyses for mutation numbers were performed using non-paired two-tailed Student's t test.

FIG. 3. Anti.gp140 Mapping by ELISA.

A) Pie charts show relative distribution of anti-gp120 and anti-gp41 antibodies among all anti-gp140 antibodies cloned from patients 1-4 (see also FIG. 6 an FIGS. 15-22 for additional patients). B) Pie charts show relative distribution of antibodies binding to gp120, gp120^core, gp120^1420R but not to gp120^D368R (CD4bs), gp120, gp120^D368R but not to gp120^1420R (C04i), gp120, gp120^D368R, gp120^1420R but not gp120^core (VL), and gp120, gp120^D368R, gp120^1420R and gp120^core (Core). C) Representative ELISA results for binding to gp140, gp120, gp41, gp120^core, gp120^D368R, and gp120^1420R. Solid lines show 447-52D (anti-VL, {Gorny, 1992}), 2F5 (anti-gp41, {Buchacher, 1994}), b12 (anti-CD4gs, {Burton, 1991}), neg. control antibody mGO53 {Wardemann, 2003}, 4-221 (anti-Core), dashed lines show 2-59 (anti-VL), 3-384 (anti-gp41), 2-1262 (anti-CD4bs). D) Competition ELISA for reactivity with gp120. b12, 1-64 anti-CD4bs, 2-491 anti-Core, 1-182 anti-CD41, and 1-79 anti-V3L were biotin labeled. Inhibition of binding 10 gp120 was measured by ELISA in competition experiments with unlabeled antibodies from patient 1, 2, 3, and 4. Each dot indicates the IC50 for an individual antibody (see also Supplementary Table 4 for the exact concentrations and IC50s). The arrows show the self-inhibitory activity of the biotinylated antibody. Non-biotinylated b12 is indicated as black, 447-52D as open circle. The blocking antibodies are grouped according to their epitopes. The IC50 for unlabeled antibody that corresponds precisely to the biotin labeled indicator was 5 μg/ml for b12, 4.6 μg/ml for 1-64, 5.4 μg/ml for 2-491, 9.2 μg/ml for 1-182, and 7.5 μg/ml for 1-79 (each indicated with arrows). Antibodies at the top of each graph did not inhibit binding.

FIG. 4. Neutralizing Activity in TZM•bl Cells.

Source of antibodies from patients 1-4 is indicated at the top. A) Pie charts show neutralizing antibodies in color and non-neutralizers in grey as well as the epitopes they recognize. The size of the slice is proportional to the clone size (see also FIGS. 15-20). Number in the center indicates the total number of tested antibodies. B) Graphs show neutralizing IC50 in μg/ml of individual antibodies specific for gp41, CD4bs, CD4i, gp120^core, VLs as indicated. The dots in the graphs correspond to the pie charts above. C) Neutralizing activity of all of the pooled anti-gp 140 antibodies (pool), irrespective of their individual neutralizing activity (top). D) Neutralizing activity of purified serum IgG from each of the patients. In b, c, and d, Y axis shows the antibody concentration in μg/ml required to achieve IC50. The numbers on the X axis represent individual viruses all of which are Clade-B unless otherwise stated; For patients 1, 3, 4: 1. MW965.23 (Clade C tier-1), 2. DJ263.8 (Clade A tier-1), 3. SF162.LS (tier-1), 4. SS1196.1 (tier-1), 5. Bal.26 (tier-1), 6. 6535.3 (tier-2). 7. RHPA4259.7 (tier-2), 8. TR0.11 (tier-2), 9. SC422661.8 (tier-2), 10. PVO.4 (tier-2). For patient2: 1. MW965.23 (Clade C tier-1), 2. DJ263.8 (Clade A tier-1), 3. SFI62.LS (tier-1), 4. SS1196.1 (tier-1), 5. Bal.26 (tier-1), 6. 6535.3 (tier-2), 7. RHPA4259.7 (tier-2), 8. CAAN5342.A2 (tier-2), 9. THRO4156.18 (tier-2), 10. SC422661.8 (tier-2) (see also FIG. 25 for IC50s for individual antibodies and pools).

FIG. 5. Neutralizing Activity of Patient Serum in TZM•bl Assays.

A) Table shows serum dilution IC50 for all six patients on selected Clade-B tier-1 and tier-2 viruses. B) Table shows serum dilution IC50s for all six patients on selected clade A/C tier-2 viruses. C) The graph summarizes each patients' tier-2 serologic activity. The Y axis shows the serum dilution IC50, each virus is represented by a different colored symbol (right), and the X axis indicates the source of the serum.

FIG. 6. Anti-gp140 Antibody Cloning.

A) Dot plots show gp140 biotin and anti-CD19 staining on blood mononuclear cells pre-gated for IgG and CD19 expression from patients 5 and 6 (pt 5 and 6) and healthy controls (hc 1 and 2). See FIG. 14 for participant profiles. Healthy controls (hc 1 and 2) were healthy HIV negative men with no known medical problems and normal blood counts. B) Pie charts show the distribution of gp140 binders and non-binders among all antibodies cloned. The number in the center indicates total number of antibodies analyzed, each pie slice represents a clonal family and the size of the slice is proportional to the number of clonal members better: relatives. Each clonal family is represented by the same shade throughout and unique antibodies that are not members of clones are not shaded. C) Dot plots show gp140 and CD19 staining of blood mononuclear cells from patient 2 and 3 as in FIG. 1. Gated non-gp140-binding cells were sorted as negative control (See also FIGS. 20 and 21).

FIG. 7. Sample Mutational Trees Showing Clonal Relationships Between Members of gp140 Binding Antibodies.

Clones showed identical IgH and IgL chain rearrangements with variations in somatic mutations as indicated by individual circles. Each clone is represented separately. The size of the circle is proportional to the number of clone members with identical somatic mutation patterns. The name of the clone members that are part of a given branch is indicated in the center of each circle. Blank circles represent uncloned intermediates.

FIG. 8. Somatic Hypermutation.

Graphs show numbers of mutations per antibody for V_H (left) grouped by patient. Vκ (middle) grouped by patient, and V_H (right) for all antibodies to a specific epitope as indicated. Stars indicate P values ≦0.001. P values were calculated by comparison to the pool of gp140 non-reactive antibodies except those below the lines, which refer to the paired samples. Each antibody clone is represented once by a single randomly selected clone member. Statistical analyses for mutation numbers were performed using non-paired two tailed Student's t test.

FIG. 9. Surface Plasmon Resonance Measurements for Interaction Between Selected Antibodies and gp140.

Graphs show antibody dissociation curves over time. The starting concentration of gp140 was 25-50 μg/ml and the different curves represent 1:2 dilutions of the starting material. The X-axis shows time in seconds and the Y-axis shows the response rate. The antibodies are indicated above each graph. Squares indicate anti-CD4bs, circles anti-gp120^core, triangle (point down) anti-CD4i and triangle (point up) anti-VL antibodies.

FIG. 10. Effect of Deglycosylation on gp120 Binding.

A) Gel electrophoresis and Coomassie blue staining or Western blot with LCA and DCA lectin of gp120 and aglyco-gp120. B) Representative ELISA results comparing binding to gp120 and aglyco-gp120 for two antibodies that are sensitive (3-42) to or not-sensitive to (3-133) gp120 deglycosylation. Lines indicate gp120 binding and aglyco-gp120 reactivity. Antibody concentration is shown on the X-axis and OD₄₀₅ on the Y axis. C) Binding to gp120 (red) or aglyco-gp120 as measured by ELISA under saturation conditions for all neutralizing antibodies from patients 1-4. Epitopes, patient source, antibody number and relative (see above) OD₄₀₅ are indicated. Stars show antibodies sensitive to deglycosylation. D) as in c, except for a selected group of gp120^core non-neutralizers. Deglycosylation was accomplished by treating 150 ug of GP120 with PNGase F (New England Biolabs) and O-glycosidase (QA Bio) in 50 mM sodium phosphate without denaturing agents at 37° C. overnight. For lectin blotting 10 ug of protein was resolved on an SDS-PAGE gel under non-reducing conditions, transferred to polyvinylidene difluoride membranes, blocked with Western Blocking Reagent (Roche), and incubated with biotinylated Lens culinaris agglutinin (Le A, 15 ug/ml, Vector Laboratories) to detect N-linked glycans or Datura stramonium lectin (DSA, 5 ug/ml, Vector Laboratories) to detect N- and O-linked glycans. The membrane was next incubated with alkaline phosphatase-conjugated goat anti-biotin antibody, and visualized with 4-nitro bluetetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate (Roche).

FIG. 10: Serum Absorption by YU2-gp140 Trimer and Binding to Control Monoclonal Antibodies.

a, ELISA results of serum IgG tested for binding to gp140, gp120 and gp41 before and after absorption with YU2 gp140 trimer. Patients 1-4 are represented in red, green, brown and blue respectively. b, binding of b12, 2G12, 447-52d, 2F5, and 4E10 to trimerized gp140.

FIG. 11: Effect of Deglycosylation on BAL gp120 Binding.

a, Gel electrophoresis and Coomassie blue staining or Western blot with LCA and DCA lectin of BAL gp120 and aglyco-BAL-gp120. b, Representative ELISA results comparing binding to gp120 and aglyco-gp120 for control antibody 2g12⁶⁷. The red line indicates gp120 binding and green lines indicate aglyco-gp120 reactivity. Antibody concentration is shown on the X axis and OD₄₀₅ on the Y axis. c, Binding to gp120 (red) or aglyco-gp120 (green) as measured by ELISA under saturation conditions for all neutralizing antibodies from patients 1-4. Epitopes, patient source, antibody number and relative (see above) OD₄₀₅ are indicated. Stars show antibodies sensitive to deglycosylation. Deglycosylation was accomplished by treating 150 μg of gp120 with PNGase F (New England Biolabs) and O-glycosidase (QA Bio) in 50 mM sodium phosphate without denaturing agents at 37° C. overnight. For lectin blotting 10 μg of protein was resolved on an SDS-PAGE gel under non-reducing conditions, transferred to polyvinylidene difluoride membranes, blocked with Western Blocking Reagent (Roche), and incubated with biotinylated Lens culinaris agglutinin (LCA, 15 μg/ml, Vector Laboratories) to detect N-linked glycans or Datura stramonium lectin (DSA, 5 μg/ml, Vector Laboratories) to detect N- and O-linked glycans. The membrane was next incubated with alkaline phosphatase-conjugated goat anti-biotin antibody, and visualized with 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Roche).

FIG. 12. Binding of Anti-gp120^core Anti-CD4Bs Antibodies to gp120 Core and Stabilized Core.

Graphs show the ELISAs for binding to YU2 gp120^core(a), and the 8b mutant YU2 gp120 core that was stabilized in the CD4 bound state⁵⁴ (b). AntiCD4bs and anti-Core antibodies from patients 1-6 were tested for binding to both forms of gp120 at a starting concentration of 100 g/ml. Control antibody b12 is shown⁶⁵, negative control antibody mgo53⁷⁰.

FIG. 13. Binding of anti-gp120^core antibodies to gp120, gp120^D368R and gp120^368/370AA.

Anti-Core antibodies from patients 1-4 and 6 were tested in ELISA for their binding to YU2 gp120, gp120^D368R and gp120^{368/370AA45-48}. Lines indicate binding to gp120, to gp120^D368R and to gp120^368/370AA. B12 is shown as a control.

FIG. 14. Patient and Control Information.

A table showing the clinical information of patients providing sera for the methods disclosed herein and clinical status.

FIG. 15. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 1.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 16. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 2.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 17. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 3.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 18. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 4.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 19. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 4.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 20. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 5 and 6.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140-reactive IgG B cells form patients 1-6.

FIG. 21. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 2.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140 non-reactive IgG B cells form patients 2 and 3.

FIG. 22. Repertoire and Reactivity of gp140 Binding Antibodies, Patient 2.

A table showing IgH and IgL chain gene sequence information and antibody reactivity and neutralization assay results of cloned antibodies. ND indicates not determined. The number of clone members with 100% IgH and IgL chain gene sequence homology is indicated and clonal relatives with various degrees of somatic mutations are shown. Antibodies from gp140 non-reactive IgG B cells form patients 2 and 3.

FIG. 23. Affinity Measurements of b12 and Selected Antibodies by Surface Plamon Resonance.

Stars indicated cases in which the sensorgrams are virtually flat during the dissociation phase. Off-rates might therefore be even slower than the ones listed. Epitopes against which the affinities were measured are indicated above. (M=Mol/liter; s=seconds)

FIG. 24. Summary of gp140 Competition ELISA Experiments.

Numbers indicate IC50s for the specific antibody in ELISA assays measuring the blocking of the binding of the indicated biotinylated antibody (indicated at right) to gp120. a, anti-gp120 core antibodies. B, anti-CD4bs antibodies. c, anti-CD4i antibodies. d, anti-VL antibodies. Biotinylated antibodies are: b12, 1-64 anti-CD4bs, 2-491 anti-Core, 1-182 anti-CD41, and 1-79 antiV3L.

FIG. 25. In Vitro Tzm-Bl Neutralization Assay.

Showing tables a and b whereby Numbers indicate IC50s for the specific monoclonal antibody, serum IgG or pooled antibodies in the Tzm-bl assay measuring inhibition of infection by the indicated viral strains. X indicates activity that did not reach IC50 values at the concentration tested for patients 1-4 and previously described control antibodies.

FIG. 26. In Vitro Tzm-Bl Neutralization Assay.

Showing tables c and d whereby Numbers indicate IC50s for the specific monoclonal antibody, serum IgG or pooled antibodies in the Tzm-bl assay measuring inhibition of infection by the indicated viral strains. X indicates activity that did not reach IC50 values at the concentration tested for patients 1-4 and previously described control antibodies.

FIG. 27. In Vitro Tzm-Bl Neutralization Assay.

Showing tables e whereby Numbers indicate IC50s for the specific monoclonal antibody, serum IgG or pooled antibodies in the Tzm-bl assay measuring inhibition of infection by the indicated viral strains. X indicates activity that did not reach IC50 values at the concentration tested for patients 5 and 6.

FIGS. 28A, 28B, 28C, 28D. Neutralization Screen of Plasma Samples Against Standard Virus Panel.

Screen of 1818 plasma samples from a cohort of HIV-1 infected elite controllers against a standard panel of HIV isolates. Shown are the reciprocal dilutions needed to achieve a 50% inhibition in the TZM•bl neutralization assay. Samples were tested primary at a 1:20 dilution and then titrated 3-fold seven times in duplicate wells. HIVIG was used as appositive control at a starting concentration of 2500 μg/ml. Negative controls were normal naïve human plasma and Murine Leukemia pseudovirus. Patients 2, 3, and 5 correspond to CTR118, CTR34 and CTR207 respectively. Patients with broad activity against tier-2 viruses are shaded.

FIG. 29. Mapping of the Core Epitope.

(A) Bar diagram shows the percental apparent binding of anti-core antibodies and b12 to mutant gp120 (D474A, M475A, R476A) relative to gp120 wildtype. Relative binding below 60% was considered to be significant decrease. (B) Ribbon diagram of gp120 (PDB ID: 3DNO {Liu J, 2008 #45}) that shows the CD4bs, the CD4is and the defined core epitope. (C) Venn diagram summarizes the sensitivity for anti-core antibodies to bind to D474A, M475A and R476A. E.g., two anti-core antibodies showed an insensitive binding to any of the three mutants, whereas 16 antibodies were sensitive for D474A and R476A.

FIG. 30. Peptide ELISA.

(A) Anti-core antibodies (black lines) do not bind the core region peptide. (B) Positive control V3-loop peptide was recognized by an anti-V3-loop antibody (2-59, {Scheid JF, 2009 #25}) (inclining line).

FIGS. 31A-E. TZMbl Neutralization Data.

(A) Values represent IC₅₀ s in μg/ml for anti-core antibodies and b12 in an TZMbl-based neutralization assay. Values in red show inhibition at the concentrations tested. X indicates that this given antibody almost reached an IC₅₀ at the highest concentration tested {Scheid JF, 2009 #25}. (B-E) Numbers indicate the apparent binding [%] of the anti-core antibodies to the different gp120 mutant proteins. Shaded fields indicate a significant decrease (below 60%) in binding. Bolded text highlight differences in binding properties compared to anti-CD4bs antibody b12.

DETAILED DESCRIPTION

Long-lived memory antibody responses are a key feature of successful immune responses and the basis of many vaccines. This type of memory resides in circulating post germinal center memory B cells and in long-lived plasma cells {Zinkernagel, 1996; Maruyama, 2000; Maclennan, 2000; Radbruch, 2006}. Antigen-specific memory B cells are rare, non-cycling cells that do not require stimulation by antigen in order to persist for long periods of time {Schittek, 1990; Maruyama, 2000}. However, they expand rapidly in response to antigen and develop into plasma cells that reside in the bone marrow and produce large quantities of antibodies for prolonged periods of time {Manz, 1997; Slifka, 1998; Radbruch, 2006}.

Provided herein are HIV neutralizing antibodies, or antigen binding portions thereof, which comprises a binding region that binds to an antigenic epitope on gp120, or a portion of the antigenic epitope, wherein the antigenic epitope is on the same face of gp120 as a CD4 binding site or on the same face as the binding site for a b12 antibody. The antigenic epitope comprises gp120^core, also the antigenic epitope comprises conformational epitope on gp120 within the α5-helix. The invention further comprises in other embodiments a vaccine comprising at least one antibody comprising gp120^core and a pharmaceutically acceptable carrier.

In yet another embodiment, a method of inhibiting virus replication or spread to additional host cells or tissues comprising contacting a mammalian cell with the antibody, or a portion thereof, as disclosed herein which binds to an antigenic epitope on gp120.

In yet another embodiment, a method for treating a mammal with infected with a virus infection comprising administering to said mammal a pharmaceutical composition comprising the anti-gp120^core antibodies disclosed herein. In yet another embodiment, the method provides for the vaccination against HIV comprising administering to a subject the vaccine disclosed herein.

A method for isolating virus neutralizing antibodies comprising: providing a viral surface protein; binding of memory B-cells to said viral surface protein; producing antibodies by said memory B-cells; and isolating antibodies and further comprising providing an artificially trimerized gp140 protein; purifying gp140 binding B-cells; and isolating antibodies. The antibody produced thereby binds to an antigenic epitope comprising gp120^core, or a portion thereof.

Antibodies can be protective against initial HIV infection in passive transfer experiments in non-human primates and can modulate viral load during infection {Mascola, 2000; Shibata, 1999; Veazey, 2003; Parren, 2001; Mascola, 1999; Trkola, 2005; Wei, 2003; Frost, 2005}. Based on these observations, it has been proposed that such antibodies may be important components of a preventative vaccine {Burton, 2004; Mascola, 2007; Karlsson Hedestam, 2008; McMichael, 2006; Zolla-Pazner, 2004}.

The present invention provides for antibodies, either alone or in combination with other antibodies, having broad neutralizing activity in serum. Neutralization activity can be the result of a single highly effective antibody such as gp120^core of the present invention, or a plurality of antibodies as described. Broadly neutralizing serological activity can be elicited by a combination of antibodies that phenocopies the natural anti-HIV immune response in patients as an effective means of protection against a large number of HIV strains.

It is an embodiment of the invention to provide for HIV neutralizing antibodies comprising at least one of an antibody, or antigen binding portion thereof, which comprises a binding region binds to an antigenic epitope on gp120, or a portion of the antigenic epitope, wherein the antigenic epitope is on the same face of gp120 as a CD4 binding site. The antigenic epitope may also be on the same face as the binding site for a b12 antibody. An aspect of the invention provides for antibodies, or an antigen binding portion thereof, which binds to a new the antigenic epitope comprises gp120^core, which binds to the same face of gp120 as b12 and CD4.

Another embodiment of the invention provides for an antibody, or antigen binding portion thereof, comprising a binding region which binds to a CDR3 region comprising at least one of SEQ ID NOs: 1-630, or fragments or derivatives thereof. The antibodies which bind to a CDR3 region further may bind to epitopes, or portions of epitopes, comprising V3, gp41, VL, CD4i or CD4bs.

In yet another embodiment, the invention provides for a vaccine comprising at least one antibody of the invention and a pharmaceutically acceptable carrier. It is a further embodiment of the invention to provide for a vaccine comprising at least one antibody described herein and a pharmaceutically acceptable carrier. The vaccine can include a plurality of the antibodies having the characteristics described herein in any combination and can further include antibodies neutralizing to HIV as are known in the art.

In another embodiment of the invention a method of inhibiting virus replication or spread to additional host cells or tissues comprising contacting a mammalian cell with an antibody, or a portion thereof, which binds to an antigenic epitope on gp120 is provided. As described the antibody or antibodies of the invention bind to the gp120^core epitope which can be found on a separate binding site on the same face of gp120 as a CD4 binding site or a b12 antibody.

The method may further comprise administering to a cell one or more antibodies which bind to a CDR3 region comprising any one of SEQ ID NOs: 1-630, or a fragments thereof.

In a further embodiment, the invention provides for a method for treating a mammal with infected with a virus infection comprising administering to said mammal a pharmaceutical composition comprising an antibody, or a portion thereof, which binds to an antigenic epitope on gp120 is provided. As described the antibody or antibodies of the invention bind to the gp120^core epitope which can be found on a separate binding site on the same face of gp120 as a CD4 binding site or a b12 antibody. The method may further comprise administering to the mammal one or more antibodies which bind to a CDR3 region comprising any one of SEQ ID NOs: 1-630, or a fragments thereof. The compositions and vaccines of the invention can include more than one antibody having the characteristics disclosed (e.g. a plurality or pool of antibodies). It may also include other HIV neutralizing antibodies as are known in the art.

It is yet another embodiment of the invention to provide for a method for vaccinating a mammal from HIV infection. The vaccines or antibody pharmaceutical compositions of this invention may be administered alone or in combination with other HIV antigens, and in one or several immunization doses.

The present invention also provides for methods of producing and isolating virus neutralizing antibodies comprising: providing a viral surface protein; binding of memory B-cells to said viral surface protein; producing antibodies by said memory B-cells; and isolating antibodies.

For isolating HIV neutralizing antibodies the method further includes providing an artificially trimerized gp140 protein; purifying gp140 binding B-cells; and isolating antibodies. An antibody, or antigen binding portion thereof, produced by the method comprising providing an artificially trimerized gp140 protein; binding of said trimerized gp140 to B-cells; purifying gp140 binding B-cells; and isolating antibodies produced by the memory B-cells. It is yet another embodiment of the invention to provide for antibodies produced by the methods disclosed.

The above described antibodies and antibody compositions or vaccine compositions, comprising at least one or a combination of the antibodies described herein, may be administered for the prophylactic and therapeutic treatment of HIV viral infection, as well as for other reasons described herein.

A “cell” can be any cell, and, preferably, is of a eukaryotic, multicellular species (e.g., as opposed to a unicellular yeast cell), and, even more preferably, is a mammalian, e.g., human cell.

A cell can be present as a single entity, or can be part of a larger collection of cells. Such a “larger collection of cells” can comprise, for instance, a cell culture (either mixed or pure), a tissue (e.g., endothelial, epithelial, mucosa or other tissue), an organ (e.g., lung, liver, muscle and other organs), an organ system (e.g., circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or an organism (e.g., a bird, mammal, or the like). Preferably, the organs/tissues/cells being targeted are of the circulatory system (e.g., including, but not limited to blood, including white blood cells). The target cells need not be normal cells and can be diseased cells. Such diseases cells can be, but are not limited to, HIV infected cells.

In one embodiment, the invention provides for a method for inducing immune responses in a subject said method comprising administering a pharmaceutical composition, including vaccines or antibody compositions, comprising HIV neutralizing antibodies disclosed herein. Such induced immune response include cellular and humoral responses.

It is to be understood that compositions may a single or a combination of antibodies disclosed herein, which may be the same or different, in order to prophylactically or therapeutically treat the progression of various subtypes of human immunodeficiency virus infection after vaccination. Such combinations may be selected according to the desired immunity. When a an antibody vaccine is administered to an animal or a human, it can be combined with one or more pharmaceutical acceptable carriers, excipients or adjuvants as are known to one of ordinary skilled in the art. The composition may further included broadly neutralizing antibodies known in the art, including but not limited to, b12, 2F5, 4E10 and 2G12.

As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” may include a combination of two or more cells; reference to “DNA” may include mixtures of DNA, and the like.

Vaccine formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection solutions and suspensions may be prepared from purified nucleic acid preparations for the DNA plasmid priming compounds and/or purified viral vector compounds commonly used by one of ordinary skill in the art. Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations may also include other agents commonly used by one of ordinary skill in the art.

The formulation may be administered through different routes, such as oral, including buccal and sublingual, rectal, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The vaccine may likewise be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. It is expected that from about one to about five dosages (e.g., two dosages—an initial inoculation, the prime of prime/boost, and a booster) may be required per immunization protocol. The initial prime and boost administrations may contain a quantity of antigen sufficient to induce a satisfactory immune response. An appropriate quantity of prime and boost antigen(s) to be administered is determined for any of the prime/boost protocols disclosed herein by one skilled in the art based on a variety of physical characteristics of the subject or patient, including, for example, the patient's age, body mass index (weight), gender, health, immunocompetence, and the like. Similarly, the volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 mL to 1.0 mL. Preferably a patient has a normal immune system and is not infected with human immunodeficiency virus, although the vaccine may also be administered after initial HIV infection to ameliorate disease progression, or after initial infection to treat AIDS.

Further, with respect to determining the effective level in a patient for treatment of HIV, in particular, suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy against HIV of various gene therapy protocols (Sarver et al. (1993b), supra). These models include mice, monkeys and cats. Even though these animals are not naturally susceptible to HIV disease, chimeric mice models (e.g., SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bone marrow-ablated BALB/c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver/thymus or other tissues can be infected with lentiviral vector or HIV, and employed as models for HIV pathogenesis. Similarly, the simian immune deficiency virus (SIV)/monkey model can be employed, as can the feline immune deficiency virus (FIV)/cat model. The pharmaceutical composition can contain other pharmaceuticals, in conjunction with a vector according to the invention, when used to therapeutically treat AIDS. These other pharmaceuticals can be used in their traditional fashion (i.e., as agents to treat HIV infection).

Since the discovery of HIV, numerous monoclonal antibodies to the envelope protein have been produced by random cloning of heavy and light chains in phage display libraries or by selection of antibody secreting hybridomas, but only a few highly active broad neutralizing antibodies have been obtained {Pantophlet, 2006; Burton, 2005; Mascola, 2007; Zolla-Pazner, 2004; Karlsson Hedestam, 2008}. Among these b12 anti-CD4bs {Burton, 1991}, 2F5 and 4E10 anti-gp41 {Buchacher, 1994; Zwick, 2001}, and 2GI2 anti-glycan {Trkola, 1996} antibodies have received the greatest attention because of their unique breadth and potency in vitro and in vivo. Ideally, a vaccine that induces such antibodies might be protective against HIV. However, to date, it has not been possible to re-isolate such antibodies from patients, or induce them by immunization in experimental animals {KarlssonHedestam, 2008; Zolla-Pazner, 2004; Burton, 2005; Burton, 2004; Mascola, 2007}.

The invention further provides for identifying and isolating antibodies from the memory compartment of B cells containing many different neutralizing antibodies with diverse activity. Therefore, broad serologic neutralizing activity can involve a combination of antibodies.

In embodiments discussed herein, the invention provides for a new core epitope to gp120 (gp120^core). Antibodies to gp120^core differ from B 12 and can be a very potent neutralizer in that it works as well as the CD4 binding site antibodies in neutralization studies. In this embodiment, the invention provides for a collection of antibodies that neutralize various strains and clades of HIV with different antibodies neutralizing different viruses. An embodiment of the invention is a vaccine comprising the epitopes identified by the antibodies disclosed.

The present invention provides for vaccines, either alone or in combination with other antigens, and epitopes, that elicit broad neutralizing activity in serum. Neutralization activity can be the result of a single epitope such as gp120^core of the present invention, or a plurality of epitopes identified by the antibodies as described. Broadly neutralizing serological activity can be elicited by a combination of antigens that phenocopies the natural anti-HIV immune response in patients as an effective means of protection against a large number of HIV strains.

To characterize memory antibody responses to HIV, antibodies from HIV envelope binding memory B cells cloned from six HIV infected patients with high titers of broadly neutralizing antibodies. The human B cell memory response to HIV can be composed of independent, expanded B cell clones can express high affinity neutralizing antibodies to the gp120 variable loops, the CD4 binding site, the co-receptor biding site, and to a new neutralizing epitope that is on the same face of gp120 as the CD4 binding site, gp120^core. The IgG memory B cell compartment in humans with serum neutralizing activity to HIV can thus be comprised of multiple individual clonal responses each with more limited anti-viral activity that in aggregate, therefore resulting in broad neutralizing activity. Effective vaccination to HIV may require a strategy that elicits a broad repertoire of antibodies to provide protection against the large number of HIV strains and the neutrability of the virus.

The antibodies produced by memory B cells purified from the blood of six patients with high serum titers of broadly neutralizing anti-HIV antibodies (FIG. 5). One of the patients was a non progressor, three were elite controllers, one a slow progressor, and one had been infected two years ago (Supplementary Table 1 {Walker, 2007}). Artificially trimerized gp 140 protein, which is a fusion of the gp120 and gp41 envelope proteins, can be used to identify and purify HIV specific B cells because this molecule resembles the native trimer in that it can bind to the broadly neutralizing anti-HIV antibodies {Scheid, 2009; Yang, 2000}. Small numbers of B cells that bind the gp140 trimer can readily detected in the IgG memory B cell compartment in the samples from patients but not in uninfected controls (FIG. 14 and FIG. 6). Individual gp140 binding memory B cells can also be purified by cell sorting, and Ig heavy and light chains can be cloned from single cell cDNA libraries {Tiller, 2008; Scheid J., 2009}. Ig heavy and light chain genes can be amplified from samples from each of four HIV infected individuals (FIG. 1a) and smaller numbers from two other samples (FIG. 6).

In embodiments discussed herein, the invention provides for a new core epitope to gp120 (gp120^core) Antibodies to gp120^core differ from B12 and can be a very potent neutralizer in that it works as well as the CD4 binding site antibodies in neutralization studies. Provided herein is the conserved epitope on gp120 that is frequently recognized by high affinity, neutralizing monoclonal antibodies cloned from HIV-1 infected individuals with broadly neutralizing serologic activity and low to intermediate viral loads. gp120^core epitope to D474A, M475A, R476A, thus to the outer domain/inner domain junction of gp120.

The antibodies disclosed herein bind to a conformational epitope recognized by neutralizing human monoclonal anti-gp120-^core antibodies. These antibodies target a conformational epitope on gp120 (D474A, M475A, R476A) found within the α5-helix of the molecule, that is highly conserved across different HIV-1 clades. Anti-core antibodies bind to a conformational three-dimentional structure of the linear epitope TNGTEIFRPGGGDMRDNWR (SEQ ID NO: 629) however the anti-core antibodies do not recognize the linear peptide epitope. Thus, anti-core antibodies do not recognize a linear epitope.

Addressing the induction of an immune response against the gp120 core epitope, anti-core antibodies show a higher affinity (one order of magnitude) to gp120_core than to gp120 {Scheid JF, 2009 #25}, which can be related to the absence of the variable loops 1 to 3 in gp120^core that may interfere with anti-core antibody binding.

In an embodiment, the invention provides for a collection of antibodies that neutralize various strains and clades of HIV with different antibodies neutralizing different viruses. An embodiment of the invention is a vaccine comprising the epitopes identified by the antibodies disclosed.

The present invention provides for vaccines, either alone or in combination with other antigens, and epitopes, that elicit broad neutralizing activity in serum. Neutralization activity can be the result of a vaccine comprising a single epitope or antigen such as gp120^core of the present invention, or a plurality of epitopes or antigens identified by the antibodies as described. Broadly neutralizing serological activity can be elicited by a combination of antigens or epitopes that phenocopies the natural anti-HIV immune response in patients as an effective means of protection against a large number of HIV strains. The way forward for a vaccine is to phenocopy the human as revealed by the following examples.

Examples

Characteristics of Anti-gp140 Memory Antibodies

In contrast to random antibody cloning from memory B cells {Mietzner, 2008; Tiller, 2007}, and to the antibodies isolated from B cells that did not bind to gp140 from the same subjects, many clonally related antibodies in the gp140 binding B cells can be formed (FIG. 1b and FIG. 6, FIG. 7 and FIGS. 15-22). Each clone was expanded to varying degrees ranging from 1-39 family members (FIG. 1b). The majority of the antibodies derived from gp140 binding memory cells belonged to clones with more than one member and in patient sample #2 all of the 141 antibodies were members of 22 expanded families related by somatic mutations. The finding that most of the antibodies isolated from the four more complete patients belonged to expanded clones suggested that a significant fraction of the gp140-reactive memory B cell repertoire was captured.

Individual antibodies were expressed by transfection and tested for reactivity to gp140 by ELISA. Eighty-six percent of all of the antibodies cloned from sorted gp140 binding B cells were gp140 reactive (FIG. 1b, d, and FIGS. 15-22). In contrast, none of the 51 antibodies obtained from the non-gp 140 binding memory B cells from two of the same patients were gp140 specific (FIG. 1b, d, and FIGS. 15-22). Overall, 432 individual anti-gp140 antibodies were obtained belonging to 132 different clones (FIGS. 15-22). The expanded clones of gp140 binding B cells contained antibodies that were related by mutation with a maximum number of 66 mutations separating the individual clonal relatives (FIG. 7 and FIGS. 15-22).

When compared to IgG antibodies derived from non-gp 140 binding B cells from the same patients or IgG memory B cells from historical controls {Tiller, 2007} the gp140 binding antibodies were enriched for V_HI {Huang, 2004}, Igκvs. Igλ, and Jκ2 or Jκ5 (FIGS. 1c and 2a, b and c). Individual samples showed longer or more charged IgH CDR3s but these features were not found in all of the samples (FIG. 2b). An unexpected finding was that anti-gp 140 antibodies were highly mutated when compared to non-gp140 binding antibodies from the same patient samples or randomly cloned IgG memory antibodies from historical controls (FIG. 2d). This difference in mutation frequency was found for both V_H and Vκ and was highly significant whether all antibodies were included or counted individual clones only a single time (FIG. 2d and FIG. 8). Anti-gp140 memory B cells can be highly selected post-germinal center cells skewed to Igκ and V_H1 usage. The exceptionally high level of mutation found in these antibodies may reflect chronic B cell immune responses to HIV with persistent somatic hypermutation and selection.

Epitopes Recognized by Anti-gp140 Memory Antibodies

To map the antigenic specificity of the gp140 binding antibodies performed ELISA experiments with purified gp120 and gp41 were performed. Seventy percent (70%) of the gp140 antibodies bound to gp120 and 30% were gp41 specific (FIG. 3a). Thus, the majority of the anti-gp140 antibodies in patient samples with broadly neutralizing anti-HIV antibodies are directed to gp120.

Anti-gp41 antibodies were further screened against a peptide library consisting of overlapping 15mers including the membrane proximal region that binds to two broadly neutralizing anti-gp41 monoclonal antibodies 2F5 and 4E10 {Muster, 1993; Zwick, 2001}. None of the 131 anti-gp41 antibodies assayed bound to the membrane proximal peptides and only one antibody bound to peptides corresponding to the previously reported immunodominant region of gp41 {Xu, 1991}. The majority of the gp41 antibodies produced by memory B cells in patients with broad serum neutralizing activity recognize conformational determinants and antibodies to the membrane proximal region can be difficult to detect in the gp140 trimer binding B cells despite the fact that both 2F5 and 4E10 bind to the trimer.

The specificity of the anti-gp120 binding antibodies was further mapped using a collection of mutant proteins: gp120^D368R interferes with binding to CD4 and nearly all known anti-CD4 binding site (anti-CD4bs) antibodies including b12 {Pantophlet, 2003; Olshevsky, 1990; Roben, 1994; Thali, 1991}; gp120^142R interferes with CD4 induced co-receptor binding site antibodies (anti-CD41) including 17b {Thali, 1993}; gp120^core lacks the variable loops (VLs) and interferes with anti-VL and CD4i antibodies {Kwong, 2000; Wyatt, 1998; Kwong, 1998}. Antibodies that bound to gp120, gp120^core, gp120^142OR but not to gp120^D368R were classified as CD4bs directed. Similarly, those that bound to gp120 but not to gp120^core were classified as anti-VL antibodies, and those that bound to gp120, and gp120^D368R, but not to gp120^1420R were classified as anti-CD4i antibodies. Anti-CD4bs, -CD4i, and -VL antibodies were found in all 4 of the more complete patients but their relative representation varied significantly between patients (FIG. 3b). Among all anti-gp140 antibodies anti-CD4bs made up 9%, anti-CD4i 16% and anti-VL 27% (FIG. 3b). All of these antibodies were also screened for binding to a library of overlapping 15mer peptides covering all of gp120. Only three of the anti-gp120 antibodies bound to the linear peptides and all of these bound to the region within the V3 loop that is also targeted by a previously described antibody 447-52D {Gorny, 1992}.

To examine the kinetic binding properties of the anti-CD4bs, -CD4i and -VL antibodies to gp140, surface plasmon resonance experiments were performed with gp140 trimer comparing the binding of 7 such antibodies to the b12 anti-CD4bs monoclonal (FIG. 9 and FIG. 23). The antibodies had rapid association and slow dissociation constants with Kds ranging from 10⁻⁸-10⁻¹¹ with b12 at the lower end of the spectrum with a Kd of 1.2×10⁻⁸ (FIG. 9 and FIG. 23). Thus, the IgG memory B cells obtained from humans that produce broad serum neutralizing activity expressed high affinity antibodies specific for the CD4bs, the CD4i site and the VLs and there was no single immunodominant epitope.

In addition to anti-CD4bs, -CD41, and -VL antibodies a group of antibodies that bound to gp120, gp120^core, gp120^368R, and gp120^1420R were found which are referred to as anti-gp120^core. These antibodies make up 18% of all anti-gp 140 antibodies varying between patients from about 3% to about 35% of the repertoire (FIG. 3b). Only one of the 24 anti-gp120^core antibodies was directed to an epitope that was sensitive to gp120 -, whereas 4 out of 13 neutralizing anti-CD4i tested showed sensitivity to deglycosylation (FIG. 4 and FIG. 10) and therefore the anti-gp120^core antibodies are not predominantly directed to glycosylation dependent epitopes. In addition, none of the anti-gp120^core antibodies bound to a peptide library consisting of overlapping 15mers of gp120. The affinity of the anti-gp120^core antibodies was comparable to the other antibodies as measured by surface plasmon resonance for 6 selected antibodies in this group (Kds of 2×10⁻⁸-4.8×10⁻¹⁰, FIG. 9 and FIG. 23).

To further examine the properties of the anti-gp120^core antibodies inhibition ELISA experiments were performed using biotin labeled neutralizing antibodies to the CD4bs (b12 and 1-64), or CD4i (1-68), or the V3L (1-79) or a representative member of the gp120^core specific group (2-491) {Binley, 2004} (FIG. 3d, FIG. 4, FIGS. 15-22, FIG. 26). As expected the results for the two anti-CD4bs antibodies, b12 and 1-64, were similar and both were inhibited by other neutralizing anti-CD4bs antibodies but not by CD4i or VL specific antibodies (FIG. 3d and Supplementary Table 2, 4, and 5). In contrast, neutralizing anti-CD4i, 1-182, was inhibited by all of the neutralizing anti-CD4bs antibodies, but only by 50% of the other anti-CD4i and 29% of the anti-VL antibodies (FIG. 3d and Supplementary Table 2, 4, and 5). The selected neutralizing anti-V3L antibody was strongly inhibited by the other anti-V3L antibodies and not by anti-CD4bs, anti-CD4i, or other neutralizing anti-VLs that were not V3L-NNNTRKSINIGPGRA (SEQ ID NO. 630) peptide specific (FIG. 3, FIG. 4 FIGS. 15-22, FIG. 26 and). The CD4bs may be in close proximity to the CD4i site and that the conformation of the CD4i site is dependent on the CD4bs {Lin, 2008; Thali, 1993; Wyatt, 1998; Rizzuto, 1998; Kwong, 1998}.

Anti-gp120^core antibodies resembled b12 and CD4bs antibodies in that they inhibited the binding of the selected anti-gp120^core, anti-CD4bs, and anti-CD4i, but they did not inhibit binding of the anti-V3L antibody. Conversely, the 2-491 anti-gp120^core antibody was inhibited by the other anti-gp120^core and the anti-CD4bs antibodies (FIG. 3d, FIGS. 15-22, FIG. 26 and). However, only three out of thirteen of the anti-CD4i antibodies and none of the seven anti-VL antibodies inhibited binding of the anti-gp120^core (FIG. 3d, FIGS. 15-22, FIG. 26 and). Anti-gp120^core antibodies can recognize an immunogenic epitope in the vicinity of the CD4bs and CD4i sites, but these antibodies differ from CD4, b12 and b17 because they bind gp-120 and gp120^1420R and therefore the epitope they recognize must be different from that recognized by CD4 and most CD4bs and CD4i antibodies. This group of antibodies shares some of the features of anti-CD4bs antibodies, and can recognize one or more epitopes on the conserved face of HIV gp120 that interacts with CD4 and the co-receptor.

HIV Neutralizing Activity

To examine the neutralizing activity of the memory antibodies, the ability to inhibit infection of TZM-bl cells by Env pseudovirus variants {Montefiori, 2005} including isolates from clades A, B, and C (FIG. 4 and) was measured. The panel of clade B viruses was expanded to contain viruses with different levels of resistance to known neutralizing antibodies {Monrefiori, 2005} ranging from tier-1 strains like SFI62.LS that can be easily neutralized to tier-2 strains like TRO.11 which are not neutralized even by potent broadly neutralizing anti-CD4bs antibody b12 {Li, 2005}. To determine whether there was intraclonal variation in neutralizing activity, many of the somatic variants of the anti-CD4bs antibodies were assayed (FIG. 7). Finally, purified serum IgG from the patients was assayed on the same viruses for comparison (FIG. 4, FIG. 5). The breadth of neutralizing activity and the relative sensitivity of different viral strains was similar for serum and purified IgG indicating that most of the neutralizing activity was in the IgG fraction. Purified IgG neutralized viruses from all 3 clades, but the activity was most pronounced for the more easily neutralized tier-1 HIV variants while high concentrations of serum IgG were required for the more resistant strains (FIG. 4).

Seventy-six percent (76%) of all anti-gp120s and none of the anti-gp41 clonal families showed neutralizing activity (FIG. 4 and). Consistent with this finding the anti-gp41 antibodies were the most highly somatically mutated of the anti gp140 antibodies (FIG. 2d, anti-gp41 average number of mutations 34 vs. 24 for anti-gp120 p<0.001) suggesting that B cells producing these antibodies may be persistently recruited to germinal centers and therefore did not exert significant selective pressure on the virus when compared to anti-gp120 antibodies.

All anti-CD4bs and 88% of all anti-gp120^core antibodies showed some neutralizing activity (FIG. 4). Of a total of 64 independent clonal families of neutralizing antibodies 22 were anti-gp120^core, 18 were anti-CD4bs, 16 were anti-CD41, and 8 were anti-VL including all three of anti-V3L antibodies (FIG. 4). As a group, the antibodies to the CD4bs and gp120^core showed the highest levels of broad cross-clade and intra-clade B activity with rare antibodies covering the more resistant tier-2 viruses (FIG. 4). Although the anti-V3L antibodies showed cross-clade neutralizing activity, and one of them neutralized tier-2 viruses at high concentrations, most of the anti-VL antibodies were more restricted (FIG. 4). Cross-clade neutralizing activity was also found in the anti-CD4i antibodies but only three of these antibodies showed activity against the more difficult to neutralize tier-2 viruses (FIG. 4 and FIG. 27).

Serum antibody absorption studies found that neutralization of tier-2 viruses was predominantly achieved by anti-CD4bs with a smaller variable contribution from anti-CD4i and unidentified antibodies, however, the resolution in such studies is limited and they cannot define the nature or number of antibodies to a specific site {Li, 2008; Li, 2007; Dhillon, 2007}. No case where a single monoclonal antibody or class of antibodies in memory B cells accounts for all of the neutralizing activity in serum was found (FIG. 4). Individual antibodies showed variable levels of activity against different viruses. For example in patient #1, the Clade-C virus MW965.23 was neutralized by CD4bs 1-621, gp120^core 1-705 and VL antibody 1-79, with anti-gp120^core 1-705 showing the highest activity (FIG. 4). In contrast, 1-621 did not neutralize the clade-B virus Bal.26, whereas 1•79 did and was superior to 1-705 (FIG. 4). Similarly in patient #2, the clade-A virus DJ263.8 is neutralized by anti-CD4bs 2-470, anti-VL 2-59 but not anti-VL antibody 2-1261, whereas the same anti-VL antibody neutralized the clade-B tier-2 virus RHPA4259.7, but neither 2-470 nor 2-59 reached IC50 against this virus (FIG. 4). Some degree of neutralizing activity was common among gp120 specific memory antibodies. These antibodies recognized a broad array of epitopes and neutralizing activity was heterogeneous for different viral isolates.

Memory B cells are long-lived cells and their antibodies reflect an individuals immune responses over time. Some of these cells differentiate into plasma cells that secrete antibodies but the relative contribution of any given memory B cell to the plasma cell compartment is unknown and therefore a pool of cloned memory B cell antibodies cannot be compared directly to serum. Pools of all antibodies for each individual patient were created and compared the pools to purified serum IgG for neutralization (FIG. 4 and FIG. 27). The pools contained equal concentrations of each of the anti-gp140 clones irrespective of clone size or neutralizing activity.

Purified IgGs from samples of all of the four persons studied neutralized nearly all of the tier1 viruses including clades A, B and C at concentrations ranging from 1-212 μg/ml. The corresponding pools of the recombinant antibodies were active against these viruses and in some cases neutralized viruses that were not neutralized by the serum IgG. For example purified IgG from patient 4 did not reach an IC50 against SS1196.1 or 6535.3 (tier-2), but these viruses were neutralized by recombinant antibodies 4-42, 4-8 and 4-433 and by pooled antibodies from this patient (FIG. 4 and FIG. 27).

Consistent with the more stringent requirements for tier-2 neutralization, only the pooled monoclonal antibodies from patient 1 and 4 completely and 2 and 3 partially reconstituted this type of activity (FIG. 4 and FIG. 27). In each case the activity of the pool was greater than that of any single antibody. For example, RHPA4259.7, TRO.11 and PVO.4 were neutralized by 1-1.24 mg/ml of the patient 1 pool (pool of 21 antibodies), but none of the individual antibodies were able to neutralize these viruses (FIG. 4 and FIG. 27). Similarly the TRO.11 virus was neutralized by the pooled antibodies from patient 4 at 1.4 mg/ml (pool of 50 antibodies). The memory antibody compartment contains a large mixture of anti-HIV neutralizing antibodies combinations of which can increase the breadth of neutralization activity.

Fine Mapping of Anti-Core Antibody Binding Site on gp120.

In order to map the epitope recognized by anti-core antibodies, assayed were anti-core antibodies for binding to 72 different alanine mutants of the HIV-1 envelope protein gp120 by capture ELISA. Controls included the anti-CD4bs antibody b12 and an anti-variable-loop antibody (1-79) {Burton DR, 1994 #10; Saphire EO, 2001 #11}; Scheid JF, 2009 #25}. Mutations that altered antibody binding by 40% or more compared to the wild type protein were considered positive. The mutated residues were initially spread across gp120 to cover a broad range of candidate binding sites and then refined based on initial binding results. In particular, included were residues from the variable-loop 2 (VL2), the silent face, the CD4bs, the CD4 is, the Phe 43-cavity {Kwong PD, 1998 #32}, but also residues that lie proximal or distal to these sites (FIG. S1).

Ten mutations were found that altered the binding of nearly all anti-core antibodies and b12 despite their being physically distant from the CD4bs recognized by b12. Based on their position and chemical characteristics, these residues (L288, I449, T450, L265A, 5264, C378, N262, F376, F383, F353) appear to be required to maintain structural integrity of the molecule.

As previously demonstrated, alanine substitutions in the CD4bs (E370A, D368A, D368A/E370A), or the Phe 43-cavity (F376A), or in close proximity to the CD4bs (N276A, P470A, R480A, W96A, E275A, D477A, Y384A) reduce binding by the anti-CD4bs antibody b12. In addition, found was a slight decrease in b12 binding to W400A, R273A, K350A all of which are not thought to contribute directly to the CD4bs. These mutations had little effect on anti-core antibody binding. Among the 72 mutants, three mutants were found (D474A, M475A, R476A) that inhibited the binding of anti-core antibodies, but had no significant effect on b12 (FIG. 1A). These adjacent residues are in close proximity to the CD4bs and cover a stretch from the CD4bs (D474) down to the α5 helix at the outer-domain/inner-domain junction of gp120 (M475, R476) (FIG. 1B). Among the 24 anti-core antibodies, only two were insensitive to these three mutations. Of the remaining 22, 16 were sensitive to both D474A and R476A (FIG. 1C), and in addition only eight of these showed altered binding to M475A (FIG. 1C).

Methods Summary

Participants.

Samples were derived from patients identified as elite controllers, long term non progressors and slow progressors based on previously described criteria {Walker, 2007; Deeks, 2007}. The controls were samples from uninfected patients. All work with human samples was performed in accordance with approved Institutional Review Board protocols.

Staining, Single Cell Sorting and Antibody Cloning.

Biotinylated YU2-gp 140 was produced and used for staining and sorting of single memory B cells as previously described {Yang, 2000; Schei, 2009; Wardemann, 2003; Tiller, 2008}. cDNA synthesis and amplification were performed as described previously {Wardemann, 2003}.

ELISA.

For ELISA-testing individual antigens were coated on 96 well plates overnight as described previously {Tiller, 2008}. For competition ELISAs YU2-gp120 {Kwong, 2000} coatedplates were washed and incubated with pre-mixed biotinylated antibody and inhibiting antibody. Binding of the biotinylated antibody was detected using streptavidin conjugated HRP (Serotec) and Horseradish Peroxidase Substrate Kit (Biorad).

Neutralization Screen.

Neutralization screens were performed as described {Montefiori, 2005; Li, 2005}. In brief, neutralization was detected as reduction in luciferase reporter gene expression after single round infection in Tzm-bl cells. In order to rule out unspecific antiviral activity in plasma and antibody samples SIVmac251.WY5 was used as a negative control.

Ig Gene Sequence Analysis.

Aliquots of the V_H, Vκ and Vλ chain second PCR products were sequenced and analyzed by Ig BLAST as described previously {Wardemann, 2003}.

Recombinant Antibody Production and Purification.

Monoclonal antibodies were produced by transient transfection of suspension cultured 293T cells with “293 fectin” according to the manufacturer's suggestion (Invitrogen). Supernatants from transfected cells were collected after about 4 days of culture. Recombinant protein was purified with Protein G beads (GE Healthcare) according to the manufacturer's instructions, dialysed against PBS in Slide-A-Lyzer Dialysis Cassettes (Pierce) and stored at 4° C.

Deglycosylation of gp120.

For deglycosylation 150 ug of GPI20 was treated with PNGase F (New England Biolabs) and O-glycosidase (QA Bio) in 50 mM sodium phosphate without denaturing agents and incubated overnight at 37° C. to ensue maximal deglycosylation. Lectin blots were preformed to verify glycan removal as previously described {Kaneko. 2006).

Surface Plasmon Resonance.

All experiments were performed with a Biacore T100 instrument (Hiacore, Inc) in HBS-EP+ running buffer (Biacore, Inc) at 25C. Samples were analyzed in kinetic experiments performed in duplicates. Antibodies were immobilized to the surface of CM5 chips (Biacore, Inc.) by standard amine coupling with final immobilization levels of 250 to 500 RU. For kinetic measurement gp140 {Yang, 2000} was injected through flow cells in 5 different concentrations (357-22 nM) in HBS−EP+ running buffer (Biaccre, Inc.) at a flow rate of 50 μl/min, with 2 min association and 5 min dissociation. k_d, k_a, and K_D values were calculated after subtraction of background binding to a control flow cell using Biacore T100 Evaluation software using the kinetic analysis and the 1:1 binding model. The sensor surface was regenerated between each experiment with a 30 second injection of 10 mM glycine-HCl pH 2.5 at a flow rate of 50 μl/min.

The foregoing description of some specific embodiments provides sufficient information that others can, by applying current knowledge, readily modify or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. In the drawings and the description, there have been disclosed exemplary embodiments and, although specific terms may have been employed, they are unless otherwise stated used in a generic and descriptive sense only and not for purposes of limitation, the scope of the claims therefore not being so limited. Moreover, one skilled in the art will appreciate that certain steps of the methods discussed herein may be sequenced in alternative order or steps may be combined. Therefore, it is intended that the appended claims not be limited to the particular embodiment disclosed herein.

Each of the applications and patents cited in this text, as well as each document or reference, patient or non-patient literature, cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text, either in a Reference List before the claims; or in the text itself; and, each of these documents or references (“herein-cited references”), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

Claims

1. An isolated or purified antibody, or antigen binding portion thereof, wherein said antibody binds specifically to a conformational hiv-1 gp120 α5-helix epitope comprising the amino acids D474, M475, and R476, wherein said antibody, or binding portion thereof, does not bind specifically to a linear peptide consisting of seq ID NO.: 628, and further wherein said binding is contingent upon the presence of amino acid residues L288, I449, T450, L265, S264, C378, N262, F383, and F376 in gp120, wherein said numbering scheme is based upon the prototypic hiv-1 isolate YU-2.

2. The antibody, or antigen binding portion thereof of claim 1, wherein said antibody, or antigen binding portion thereof neutralizes at least two different isolates of the hiv-1 virus selected from the group consisting of clade A, clade B, or clade C.

3. The antibody of claim 2, wherein the antibody binds to a clade A hiv-1 virus isolate, and said antibody neutralizes a titer consisting of said clade A hiv-1 virus isolate with an IC50 less than 25.

4. A method of inhibiting hiv-1 viral replication in a host comprising administering an effective amount of a composition comprising the antibody of claim 1 to said host.